PROJECT SUMMARY/ABSTRACT The long term goal of this initiative is to target novel mechanisms of GLI1 activation as the basis for the development of novel anti-cancer therapeutics. Developmental signaling pathways such as hedgehog (Hh) play an important role in the pathogenesis and progression of many cancers, with new therapeutic approaches inhibiting this pathway in advanced preclinical studies or early clinical trials. Due to its role in a wide spectrum of cancers, pharmacological inhibition of the hedgehog (Hh)/GLI pathway has therapeutic value. Despite the recent approvals of the first Hh pathway inhibitors vismodegib and sonidegib, drug resistance has been observed in the clinic due to acquired mutations in their target Smo. There is considerable evidence that GLI1, the downstream effector of Hh signaling, may provide an alternative and more widely available therapeutic target than SMO. Activation of GLI via gene amplification is linked to tumorigenesis and invasiveness. There is significant literature that targeting GLI1 overexpression either genetically or pharmacologically, reduces cancer cell growth and proliferation including studies by the PI. Nuclear expression of GLI1 is a strong predictor of poor prognosis in pancreatic cancer, breast cancer and a marker of Hh pathway activity in brain gliomas. Targeting transcription factors directly is challenging and current GLI1 antagonists are hampered by low potency, poor in vivo efficacy, and a lack of understanding of mechanism and direct target. GLI1 is activated by phosphorylation with its nuclear translocation being mediated by the kinase DYRK1A and our lab has identified the site of DYRK1A mediated phosphorylation of GLI1. Further, we have implemented a DYRK1A high throughput screen and established a testing funnel to identify inhibitors with selectivity, cellular and in vivo efficacy. We propose that GLI transcription factors and their modulators are viable therapeutic targets and hence the goals of the current proposal are to identify DYRK1A-mediated phosphorylation sites on GLI1 in cells and determine if phosphorylation correlates with nuclear translocation of GLI1 ( Aim 1). We will then assess whether genetic or pharmacological inhibition of DYRK1A blocks GLI1 activation, transcriptional activity and cancer cell growth (Aim 2). Novel DYRK1A inhibitors identified in Aim 2 and DYRK1A knockdown cells will be assessed in glioma and breast cancer 3D spheroid and xenograft models to investigate the role of DYRK1A-GLI1 signaling in tumor growth (Aim 3). Completion of the proposed Aims will implicate DYRK1A as a new tumor-selective therapeutic target for the treatment of GLI1 activated cancers. As the current clinical inhibitors exclusively target SMO, effective clinical candidates to alternative targets in the Hh pathway are of significant interest. Identifying novel DYRK1A inhibitors to block GLI1 activation could be a very powerful approach in treating cancers overexpressing GLI1.